The present invention is directed to novel molecular methods useful for quickly and unambiguously identifying small organic molecule ligands for binding to specific sites on target biological molecules. Small organic molecule ligands identified according to the methods of the present invention find use, for example, as novel therapeutic drug lead compounds, enzyme inhibitors, labeling compounds, diagnostic reagents, affinity reagents for protein purification, and the like.
The primary task in the initial phase of generating novel biological effector molecules is to identify and characterize one or more tightly binding ligand(s) for a given biological target molecule. In this regard, many molecular techniques have been developed and are currently being employed for identifying novel ligands that bind to specific sites on biomolecular targets, such as proteins, nucleic acids, carbohydrates, nucleoproteins, glycoproteins and glycolipids. Many of these techniques exploit the inherent advantages of molecular diversity by employing combinatorial libraries of potential ligand compounds in an effort to speed up the identification of functional ligands. For example, combinatorial synthesis of both oligomeric and non-oligomeric libraries of diverse compounds combined with high-throughput screening assays has already provided a useful format for the identification of new lead compounds for binding to chosen molecular targets.
While combinatorial approaches for identifying biological effector molecules have proven useful in certain applications, these approaches also have some significant disadvantages. For example, current synthetic technology is limited in that it allows one to synthesize only a relatively small fraction of the total number of possible library members for any given molecule type. As such, even when appropriate high-throughput screening assays are available for screening a library, only a small fraction of the total number of possible members of any molecule type will be represented in the library and, therefore, -screened for the ability to bind to the chosen target. Thus, combinatorial approaches often do not allow one to identify the xe2x80x9cbestxe2x80x9d ligand for a target molecule of interest.
Additionally, even when appropriate screening assays are available, in many cases techniques which allow identification of the actual library member(s) which bind most effectively to the target are not available or provide ambiguous results, making the actual identification and characterization of functional ligand molecules difficult or impossible. Furthermore, many approaches currently employed to identify novel ligands are dependent upon only a single specific chemistry, thereby limiting the usefulness of such approaches to only a narrow range of applications. Finally, many of the approaches currently employed are expensive and extremely time-consuming. Thus, there is a significant interest in developing new methods which allow rapid, efficient and unambiguous identification of small organic molecule ligands for selected biomolecular targets. It is also desired that such techniques are adaptable to a variety of different chemistries, thereby being useful for a wide range of applications.
Schiff base adduct formation involves the reaction of an available aldehyde or ketone functionality with a primary amine to form an imine-bonded complex. While the Schiff base adduct is relatively unstable, numerous groups have employed aldehyde or ketone compounds for bonding to primary amine functionalities on proteins of interest for a Variety of purposes (see, e.g., Pollack et al., Science 242:1038-1040 (1988), Abraham et al., Biochemistry 34:15006-15020 (1995) and Boyiri et al., Biochemistry 34:15021-15036 (1995)). We herein describe novel techniques useful for rapidly and efficiently identifying organic molecule ligands that bind to specific sites on biomolecular targets, wherein those techniques are adaptable to a variety of different chemistries, preferably Schiff base adduct formation between a target polypeptide and one or more members of a library of potential organic molecule ligands. These methods allow one to unambiguously identify and characterize the organic molecule ligand that binds most efficiently to the chosen target. Additionally, the herein described methods are quick, easy to perform and inexpensive as compared to other currently employed methods.
Applicants herein describe a molecular approach for rapidly and efficiently identifying small organic molecule ligands that are capable of interacting with and binding to specific sites on biological target molecules, wherein ligand compounds identified by the subject methods may find use, for example, as new small molecule drug leads, enzyme inhibitors, labeling compounds, diagnostic reagents, affinity reagents for protein purification, and the like. The herein described approaches allow one to quickly screen a library of small organic compounds to unambiguously identify those that have affinity for a particular site on a biomolecular target. Those exhibiting affinity for interacting with a particular site are capable of forming a covalent bond with a chemically reactive group at that site, whereby small organic compounds capable of covalent bond formation may be readily identified and characterized. Such methods may be performed quickly, easily and inexpensively and provide for unambiguous results. The small organic molecule ligands identified by the methods described herein may themselves be employed for numerous applications, or may be coupled together in a variety of different combinations using one or more linker elements to provide novel binding molecules.
With regard to the above, one embodiment of the present invention is directed to a method for identifying an organic molecule ligand that binds to a site of interest on a biological target molecule, wherein the method comprises:
(a) obtaining a biological target molecule that comprises or has been modified to comprise a chemically reactive group, wherein the site of interest on the target molecule comprises the chemically reactive group;
(b) combining the target molecule with one or more members of a library of organic compounds that are capable of covalently bonding to the chemically reactive group, wherein at least one member of the library binds to the site of interest to form a covalent bond with the chemically reactive group to form a target molecule/organic compound conjugate; and
(c) identifying the organic compound that forms a covalent bond with the chemically reactive group.
In particular embodiments, the biological target molecule is a polypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, a glycopeptide or a glycolipid, preferably a polypeptide, which may be, for example, an enzyme, a hormone, a transcription factor, a receptor, a ligand for a receptor, a growth factor, an immunoglobulin, a steroid receptor, a nuclear protein, a signal transduction component, an allosteric enzyme regulator, and the like. The target molecule may comprise the chemically reactive group without prior modification of the target molecule or may be modified to comprise the chemically reactive group, for example, when a compound comprising the chemically reactive group is bound to the target molecule.
Other embodiments of the above described methods employ libraries of organic compounds which comprise aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, thioesters, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds and/or acid chlorides, preferably aldehydes, ketones, primary amines, secondary amines, alcohols, thioesters, disulfides, carboxylic acids, acetals, anilines, diols, amino alcohols and/or epoxides, most preferably aldehydes, ketones, primary amines, secondary amines and/or disulfides.
Methods for identifying the organic compound that forms a covalent bond with the chemically reactive group on the target molecule may optionally include processes that employ mass spectrometry, high performance liquid chromatography and/or fragmenting the target protein/organic compound conjugate into two or more fragments.
A particularly preferred embodiment of the present invention is directed to a method for identifying an organic molecule ligand that binds to a biological target molecule of interest, wherein the method comprises:
(a) obtaining a biological target molecule that comprises or has been modified to comprise a first reactive functionality,
(b) reacting the target molecule with a compound that comprises (1) a second reactive functionality and (2) a chemically reactive group, wherein the second reactive functionality reacts with the first reactive functionality of the target molecule to form a covalent bond, thereby providing a target molecule comprising the chemically reactive group linked to the target molecule through a covalent bond;
(c) combining the target molecule with one or more members of a library of organic compounds that are capable of covalently bonding to the chemically reactive group, wherein at least one member of the library forms a covalent bond with the chemically reactive group to form a target molecule/organic compound conjugate; and
(d) identifying the organic compound that forms a covalent bond with the chemically reactive group.
Preferably, the covalent bond formed from reaction of the first and second reactive functionalities is a disulfide bond formed between two thiol groups and optionally, subsequent to step (c) and prior to step (d) one may liberate the covalently-bonded organic compounds from the conjugate by treatment with an agent that disrupts the disulfide bond, wherein that agent may comprise, for example, dithiothreitol, dithioerythritol, xcex2-mercaptoethanol, sodium borohydride or a phosphine, such as tris-(2-carboxyethyl)-phosphine (TCEP). In various embodiments, the biological target molecule is as described above, preferably a polypeptide that may be obtained, for example, as a recombinant expression product or synthetically The thiol group and thiol functionality may be masked or activated
In particularly preferred embodiments, the chemically reactive group is a primary or secondary amine group and the library of organic compounds comprises aldehydes and/or ketones, preferably aldehydes, or the chemically reactive group is an aldehyde or ketone group, preferably an aldehyde, and the library of organic compounds comprises primary and/or secondary amines, thereby allowing Schiff base adduct formation between the chemically reactive group and members of the library. Subsequent to Schiff base adduct formation but prior to identifying the covalently-bound organic compound, a reducing agent may optionally be employed to reduce the imine bond of the Schiff base adduct.
Yet another embodiment of the present invention is directed to a method for identifying a ligand that binds to a biological target molecule of interest, wherein the method comprises:
(a) identifying a first organic molecule ligand that binds to the biological target molecule by at least one of the methods described above;
(b) identifying a second organic molecule ligand that binds to the biological target molecule by at least one of the methods described above; and
(c) linking the first and second identified organic molecule ligands through a linker element to form a conjugate molecule that binds to the target molecule.
Preferably, the biological target molecule is a polypeptide. In certain embodiments, the first and second organic molecule ligands may bind to the same site on the target molecule or to different sites thereon. The first and second organic molecule ligands may also be from the same or from different chemical classes.
Additional embodiments of the present invention will become evident to the ordinarily skilled artisan upon review of the present specification.